1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns the elucidation and uses of de-sentrinase (xe2x80x9cSENP1xe2x80x9d).
2. Description of Related Art
Sentrinization, a protein modification pathway present in all eukaryotic cells, is essential for cellular viability. The inventors have elucidated sentrin and the enzymatic mechanism required in the transfer of sentrin to its substrates (Okura et al., 1996; Kamitani et al., 1997a, b; Kamitani et al., 1998a; Kamitani et al., 1998b; Kamitani et al., 1998c; Gong et al., 1997; Gong et al., 1999). As shown in FIG. 1, sentrin is transferred through a unique E1 complex (Aos1/Uba2) and E2 enzyme (Ubc9) to covalently modify a limited subset of cellular proteins (Gong et al., 1997; Gong et al., 1999).
Sentrin-1 (also called SUMO-1) is a protein that can covalently modify specific proteins in a manner analogous to ubiquitination (Okura et al., 1996; Kamitani et al., 1997a; Kamitani et al., 1997b; Matunis et al., 1996; Mahajan et al., 1997; Boddy et al., 1996; Hershko et al., 1998). In mammalian cells, there are three known sentrin family proteins that are expressed in all tissues and appear to have overlapping function (Kamitani et al., 1997a; Kamitani et al., 1997b; Kamitani et al., 1998a; Kamitani et al., 1998b).
It is now clear that the sentrinization pathway utilizes a unique activating-enzyme complex (Uba2/Aos1) and conjugation enzyme (Ubc9), to catalyze the modification of a subset of mammalian proteins, such as PML, Sp100, RanGAP1, RanBP2, IxcexaBxcex1, Cdc3, and cytomegalovirus IE1. (Matunis et al., 1996; Mahajan et al., 1997; Kamitani et al., 1998b; Kamitani et al., 1998c; Gong et al., 1997; Gong et al., 1999; Desterro et al., 1998; Muller et al., 1998; Sternsdorf et al., 1997; Saitoh et al., 1998). Other sentrinized proteins include Dorsal, GLUT1, GLUT4, HIPK2, p53, topoisomerase I and II, Werner syndrome gene product, MDM2, and bovine papilloma virus E1 (Yeh et al., 2000; Mao et al, 2000a; Mao et al., 2000b; Buschmann et al., 2000; Ramgasamy et al., 2000). Demonstration of sentrin modification in early studies was complicated by the presence of enzymes that cleaved the isopeptide linkage between sentrin and various target proteins in the cell lysate (Kamitani et al., 1997a; Kamitani et al., 1997b; Matunis et al., 1996). Thus, in analogy to the ubiquitin pathway (Wilkinson, 1997), enzyme(s) capable of removing sentrin from sentrinized proteins must also exist.
A Saccharomyces cerevisiae enzyme, Ulp1, was recently been shown to have de-conjugating activity for Smt-3 (yeast homologue of sentrin)-conjugated proteins and is required for cell cycle progression. Ulp1 was reported by Li et al., who showed that Ulp1 can remove Smt-3, the yeast homologue of sentrin-1, from its conjugates (Li and Hochstrasser, 1999). Ubp1 also cleaves sentrin-1/SUMO-1, but not ubiquitin, from modified proteins in vitro. Ulp1 is not related to any known de-ubiquitinating enzyme. Li et al. also cited a partial EST sequence, tentatively called HsUlp1, which is homologous to yeast Ulp1.
The inventors"" invention embodies the first description of a human de-sentrinase, SENP1. The inventors have cloned a novel protease, called SENP1, which is active against sentrin, but not ubiquitin or NEDD8-modified proteins in vivo. The inventors also elucidate the genomic organization of the SENP1 gene and show that SENP1 differentially regulates sentrin-modified proteins in vivo.
The invention relates generally to compositions of and methods for obtaining de-sentrinase (SENP1) polypeptides. The invention relates as well to polynucleotides encoding SENP1 polypeptides, the recombinant vectors carrying those sequences, the recombinant host cells including either the sequences or vectors, and recombinant de-sentrinase polypeptides. By way of example, the invention discloses the cloning and functional expression of the SENP1 polypeptides. The invention includes as well, methods for using the isolated, recombinant SENP1 polypeptides in assays designed to select and improve substances capable of interacting with SENP1 polypeptides for use in diagnostic, drug design and therapeutic applications. Such substances may specifically bind to SENP1. Candidate substances that affect the activity of SENP1, such as by altering its ability to remove sentrin from sentrinized proteins may be considered modulators of SENP1.
Ulp1 is distantly related to SENP1 via sequence analysis. However, it cannot be identified through BLAST search using SENP1 polypeptide sequence. The inventors cloned the complete coding sequence of HsUlp1 and found that it did not possess protease activity against sentrin-1 conjugates. Therefore, there appears to be no functional relationship between Ulp1 and SENP1.
In some embodiments, the invention relates to isolated and purified polynucleotides or nucleic acid sequences. These nucleic acid sequences may be sequences of almost any length of nucleotides from SEQ ID NO:1, SEQ ID NO:7, or SEQ ID NO:9, or variants thereof. Such nucleic acid sequences may be identical or complementary to all or part of SEQ ID NO:1, SEQ ID NO:7, or SEQ ID NO:9. For example, these nucleic acids may encode probes, primers, truncated coding sequences, full length coding sequences, and expression constructs. These nucleic acid sequences may be comprised within genetically engineered constructs, vectors, plasmids, eukaryotic or prokaryotic host cells, or any other suitable. These nucleic acids may be DNA or RNA from a natural or synthetic source. These nucleic acids may comprise modified bases. In certain preferred embodiments, these nucleic acids will encode a peptide sequence comprising having all or a portion of the amino acid sequence of SENP1.
In other aspects, the invention relates to polypeptides comprising all or part of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8, or SEQ ID NO:10, including truncated, modified, mutated, or natural or engineered variants thereof. Methods for producing such polypeptides are described elsewhere in the application.
In some embodiments, the invention relates to the methods for preparing polypeptides comprising: transfecting a cell with a polynucleotide sufficient to produce a transformed host cell; and maintaining the transformed host cell under biological conditions sufficient for expression of the polypeptide. The polynucleotides used in this method and polypeptides obtained from this method may be any of the polynucleotides described above. In particular this method may be used to produce to polypeptides comprising all or part of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8, and SEQ ID NO: 10, including truncated, modified, mutated, or natural or engineered variants thereof. These methods may further comprise steps of purifying the obtained polynucleotides by any number of known methods described below.
These methods may also comprise steps of assaying the expressed polypeptides for activity or using the expressed polypeptides to assay any of a number of candidate substances for activity, as described elsewhere in this application. When such activity assays are done, they may be done in with purified polypeptide, polypeptides contained in a crude cell extract, or polypeptides contained in more or less intact host cells expressing the polypeptides. In more specific embodiments, the invention contemplates methods for using isolated, recombinant SENP1 polypeptides in assays designed to select and improve substances capable of interacting with SENP1 polypeptides for use in diagnostic, drug design and therapeutic applications such as anti-proliferative, anti-Herpes simplex 1, anti-CMV therapy, and other therapies as are described elsewhere in the specification and will be apparent to those of skill in the art in view of this specification.
Consistent with long-standing patent law, the words xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d denote xe2x80x9cone or more,xe2x80x9d when used in the text or claims of this specification conjunction with the word xe2x80x9ccomprisingxe2x80x9d or where the context of the usage suggests that, from either a grammatical or scientific standpoint, these words should so denote.